CD1d-restricted, invariant natural killer T (iNKT) cells which sense self and microbial lipid antigens are increasingly recognized to play a critical role i inflammatory bowel disease (IBD). The current research proposal addresses the unanswered question of how CD1d functions in specific parenchymal and hematopoietic cells in intestines to regulate iNKT cells and how this information relates to the pathogenesis and treatment of IBD. Our long-term goals are to define the specific CD1d+ cells involved in regulating or promoting iNKT cell- mediated inflammation, how exogenous microbial factors (e.g. Bacteroides fragilis sphingolipids) and endogenous factors (e.g. acid sphingomyelinase regulation of sphingomyelin levels) affect these cell-type associated CD1d functions and acquire new insights into the mechanism(s) by which CD1d mediated IL-10 production affects the capabilities of the intestinal epithelial cell (IEC) barrier through an examination of Paneth cell (PC) function; a CD1d+ IEC subset. The objective of this research is to elucidate how, at both the molecular and physiological levels, CD1d-restricted iNKT cells pathways function in homeostatic and pathologic responses. Our central hypothesis is that specific subsets of CD1d-bearing cells in intestinal tissues and the lipid antigens presented play a critical role in determining the iNKT cel activity and responsiveness to environmental agents that promote or regulate CD1d-restricted intestinal inflammation. This rationale is derived from recent insights demonstrating early life events impact significantly on later life susceptibility to environmental factors that stimulate CD1d-restricted, iNKT cell mediated intestinal inflammation. Our central hypothesis will be tested with four specific aims: 1) Determine which professional CD1d-bearing antigen presenting cell (APC) subset(s) serves a regulatory role in determining iNKT cell responses. 2) Identify the APC subset required for mediating the protective effects elicited by sphingolipids derived from B. fragilis during early life. 3) Determine the role played by sphingomyelin as an endogenous regulator of CD1d-iNKT cell function. 4) Elucidate mechanisms by which IL-10 derived from the IEC protects the barrier. In Aim 1, we will characterize the role of APC subsets in influencing CD1d-restricted responses and the inflammatory or regulatory behavior of colonic iNKT cells. Aim 2 will elucidate how early life exposure to B. fragilis-derived glycosphingolipids inhibit iNKT cell expansion and prevent CD1d-restricted inflammatory responses during later life by establishing the APC subset(s) and mechanism(s) involved. Aim 3 aims to uncover an endogenous pathway that serves to regulate iNKT cell function in later life through ASM mediated degradation of sphingomyelin. In Aim 4, we seek to understand how CD1d- restricted regulation of IL-10 extends to PC function. Overall, this proposal is significant because it will promote our understanding of how CD1d-restricted, iNKT cell pathways are affected by environmental, genetic and microbial factors and how iNKT cells in turn promote or prevent intestinal inflammation. This research will provide a firm basis upon which to develop a variety of new therapeutic strategies for IBD.